SpeckSeq enables high-throughput functional stratification of MEFV variants in autoinflammatory diseases

Abstract

Variants of uncertain significance (VUS) are a major obstacle in genetic diagnosis, particularly when involving gain-of-function (GoF) mutations that are poorly predicted in silico. MEFV, which encodes the inflammasome sensor pyrin, is mutated in two autoinflammatory diseases, familial Mediterranean fever (FMF) and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). Here, we developed SpeckSeq, a method that combines DNA bar-coding, ASC speck-based single-cell sorting and next-generation sequencing to systematically identify hypermorphic MEFV variants in response to different stimuli. SpeckSeq identified 49 GoF mutations separated into two distinct groups containing either PAAND variants or FMF variants. SpeckSeq was validated using patients' cells and supported a reclassification of MEFV variant pathogenicity, leading to novel diagnoses. As a large-scale mutagenesis approach, using human genetics as a guide, SpeckSeq revealed structural and functional pyrin features, including a putative ligand-accommodating cavity in the B30.2 domain. Altogether, SpeckSeq classifies VUS to refine molecular diagnostics and improve our knowledge on the pyrin inflammasome.